Maternal deprivation in rats is associated with corticotrophin-releasing hormone (CRH) promoter hypomethylation and enhances CRH transcriptional responses to stress in adulthood

Abstract

Exposure to stress during early development causes long-lasting alterations in behaviour and hypothalamic pituitary adrenal (HPA) axis activity, including increased expression of corticotrophin-releasing hormone (CRH). To determine whether early-life stress causes epigenetic changes in the CRH promoter leading to increased CRH transcription, 8-week old female and male rats, subjected to maternal deprivation (MD) between days 2 and 13 post-birth, were studied for HPA axis responses to stress and CRH promoter methylation in the hypothalamic paraventricular nucleus (PVN) and central nucleus of the amygdala (CeA). Plasma corticosterone and PVN CRH heteronuclear (hn)RNA responses to acute restraint stress were higher in MD rats of both sexes. DNA methylation analysis of the CRH promoter revealed a significantly lower percentage of methylation in two CpGs preceding (CpG1) and inside (CpG2) the cyclic AMP-response element (CRE) at -230 bp in the CRH promoter in the PVN but not the CeA of MD rats. Gel-shift assays, using nuclear proteins from forskolin-treated hypothalamic 4B cells and CRH promoter CRE oligonucleotides, unmethylated or methylated at CpG1, revealed a strong band that was supershifted by phospho-cAMP response element-binding antibody. This band was 50% weaker using oligonucleotides methylated at CpG2 (intra-CRE), or methylated at both CpG1 and CpG2. These findings demonstrate that HPA axis hypersensitivity caused by neonatal stress causes long-lasting enhanced CRH transcriptional activity in the PVN of both sexes. Hypomethylation of the CRH promoter CRE, a region critical for CRH transcriptional activation, could serve as a mechanism for the increased transcriptional responses to stress observed in MD rats.

Fig. 1

Effect of maternal deprivation from post-natal day 2 to 13 on body weight (A) adrenal weight (B) and thymus weight (C) measured in 60 day old male and female rats. Bars represent the mean ± S.E.M of values obtained from 9 to 11 rats per group. * P< 0.05, compared to the respective control group, following analysis by one-way-ANOVA.

Fig. 2

Effect of maternal deprivation from post-natal days 2 to 13, and restraint stress on plasma levels of ACTH (A and B) and corticosterone (C and D) in 60-day old female (A and C) and male B and D) rats. Rats were killed by decapitation in basal conditions or following 30 and 60 min restraint stress. Bars represent the average ± S.E.M of values obtained in 5 rats per group. * P< 0.001, compared to basal; # P<0.05 compared with the respective control group (two-way ANOVA).

Fig. 3

Effect of maternal deprivation from post-natal days 2 to 13, and restraint stress on CRH hnRNA levels in the hypothalamus (A and B) and central nucleus of the amygdala (CeA) (C and D) of 61-day old female (A and C) and male (C and D) rats in basal conditions or following 30 or 60 min restraint stress. Bars represent the average ± S.E.M. of values obtained in 5 rats per group. Data are expressed as absolute levels of CRH hnRNA measured by intronic qRT-PCR using a standard curve as described in Methods. * P< 0.05, compared to respective basal; #, P<0.05 compared with the respective control group (two-way ANOVA).

Fig. 4

Sequence of the proximal CRH promoter (−254 bp to −55 bp) showing the CpG dinucleotides (in red) present in this region. The numbers following each CpG identify the dinucleotides subjected to methylation analysis. TSP: Transcription start point; TBP: TATA binding protein site; GAGA: GAGA factor site; CRE: cyclic AMP response element. CRE is located at −230 bp to −222 bp. CpG1 is located just beside of CRE at −232bp and CpG2 is located inside of CRE at −226 bp.

Fig. 5

Methylation analysis of the CpG dinucleotide island within the rat CRH promoter region in the hypothalamic paraventricular nucleus of female (A) and male (B) rats. In PVN of MD rats, significantly lower methylation of CpG#1 (C) and CpG#2 (D) at −230 bp the cyclic AMP-responsive element of the CRH promoter was observed. Bars represent the average ± S.E.M of values obtained in 9 to 11 rats per group. Differences were not significant in the overall analysis by two-way-ANOVA. The pooled data for male and females for CpG1 and CpG2 are shown in C and D, respectively. * P< 0.05, MD vs controls by Student’s t test analysis.

Fig. 6

Methylation analysis of the CpG dinucleotides within the CRH promoter region in the central amygdala of female (A) and male (B) rats. Bars represent the average ± S.E.M. No significant differences were found with two-way-ANOVA or Student’s t test analysis of the data.

Fig. 7

Effect of methylation at CpGs immediately preceding (metC-1) or within (metC-2) the CRE, or methylation of both CpGs (metC1&2), on phospho-CREB (pCREB) binding to CRH promoter oligonucleotides. The sequence of the oligonucleotide is shown in A, and a representative image of a gel is shown in B. Bars represent the mean ± S.E.M of values obtained from shifted (filled bars) and super-shifted (open bars) bands in three experiments (C). *P< 0.05 compared to wild type oligonucletide (WT) (one-way ANOVA analysis).